Our laboratory investigates the molecular mechanisms by which transmembrane proteins (referred to as cargo) are sorted to different compartments of the endomembrane system in eukaryotic cells. This system comprises an array of membrane-enclosed organelles including the endoplasmic reticulum (ER), the Golgi apparatus, the trans-Golgi network (TGN), endosomes, lysosomes, lysosome-related organelles (LROs) (e.g., melanosomes), and different domains of the plasma membrane in polarized cells such as epithelial cells and neurons. Transport of cargo between these compartments is mediated by vesicular/tubular carriers that bud from a donor compartment, translocate through the cytoplasm, and eventually fuse with an acceptor compartment. Work in our laboratory focuses on the molecular machineries that mediate these processes, including (1) sorting signals and adaptor proteins that select cargo proteins for packaging into the transport carriers, (2) microtubule motors and organelle adaptors that drive movement of the transport carriers and other organelles through the cytoplasm, and (3) tethering factors that promote fusion of the transport carriers to acceptor compartments. These machineries are studied in the context of different intracellular transport pathways, including endocytosis, recycling to the plasma membrane, retrograde transport from endosomes to the TGN, biogenesis of lysosomes and LROs, and polarized sorting in epithelial cells and neurons. Knowledge gained from this basic research is applied to the elucidation of disease mechanisms, including congenital disorders of protein traffic such as the pigmentation and bleeding disorder Hermansky-Pudlak syndrome (HPS), hereditary spastic paraplegias (HSPs) and pontocerebellar hypoplasias. Lysosome Positioning Influences mTORC1 and mTORC2 Signaling This past year, we examined the role of lysosome positioning on mTOR signaling in response to growth factors. Binding of growth factors to cognate receptors at the cell surface initiates intracellular signaling cascades that eventually reach organelles such as lysosomes. A key component of these signaling cascades is the serine/threonine kinase mTOR, which exists as a subunit of two complexes named mTORC1 and mTORC2. Whereas mTORC1 associates with lysosomes, the intracellular distribution of mTORC2 is less well established. We found that perinuclear clustering of lysosomes induced by uncoupling lysosomes from kinesin motors delayed the reactivation of mTORC1 by addition of serum (a source of growth factors). This finding indicated that increasing the distance of lysosome-associated mTORC1 from the source of growth factor signaling at the plasma membrane delays the relay of signals through the cytoplasm. In addition, we made the surprising finding that mTORC2 reactivation after serum replenishment was also delayed by perinuclear clustering of lysosomes. These experiments demonstrated the existence of pools of both mTORC1 and mTORC2 that are sensitive to lysosome positioning, a finding that may explain how changes in lysosome positioning in cancer cells promote their proliferation. Reversible Association with Motor Proteins (RAMP): A Streptavidin-Based Method to Manipulate Organelle Positioning We also developed a novel method, named reversible association with motor proteins (RAMP), to manipulate organelle positioning within the cytoplasm. RAMP consists of co-expressing (i) an organellar protein fused to the streptavidin-binding peptide (SBP), and (ii) motor domains from plus-end-directed or minus-end-directed kinesin motors fused to streptavidin. The SBP-streptavidin interaction drives accumulation of organelles at the plus or minus end of microtubules, respectively. Importantly, addition of biotin dissociates the motor from the organelle, allowing restoration of normal patterns of organelle transport and distribution. We demonstrated that this method can be used to manipulate the distribution of various intracellular organelles, including lysosomes, mitochondria, peroxisomes and the endoplasmic reticulum. This method should be useful to examine how the positioning of these organelles affects their functions, and to analyze the movement of organelle cohorts upon release from the kinesin motor. A Novel Neurodevelopmental Disorder Caused by Mutations in the VPS51 Subunit of the GARP and EARP Complexes The Golgi-associated retrograde protein (GARP) and endosome-associated recycling protein (EARP) complexes are related heterotetrameric complexes that associate with the TGN and recycling endosomes, respectively. GARP and EARP function to coordinate the SNARE-dependent fusion of endosome-derived transport carriers with their corresponding compartments, enabling retrograde transport to the TGN and recycling to the plasma membrane. GARP is composed of VPS51, VPS52, VPS53 and VPS54 subunits, whereas EARP is composed of VPS50, VPS51, VPS52 and VPS53 subunits. Although these complexes are known to play key roles in intracellular protein trafficking, their importance in human physiology remains poorly understood. In collaboration with David Everman (Greenwood Genetic Center), we recently identified compound heterozygous mutations in the gene encoding the shared GARP/EARP subunit VPS51 in a 6-year-old patient with severe global developmental delay, microcephaly, hypotonia, epilepsy, pontocerebellar abnormalities, liver dysfunction, lower extremity edema and dysmorphic features. Biochemical and cellular analyses showed that the mutation in one allele causes a frameshift that produces a longer but highly unstable protein, whereas the mutation in the other allele produces a protein with a single amino acid substitution that is stable but assembles less efficiently with the other GARP/EARP subunits. These mutations consequently result in reduced levels of GARP and EARP complexes in the patients cells. In addition, the patient's cells exhibit lysosomal abnormalities, consistent with the requirement of GARP for the sorting of acid hydrolases to lysosomes. These findings thus identified a novel genetic locus for a neurodevelopmental disorder and highlighted the critical importance of GARP/EARP function in cellular and organismal physiology. ARFRP1 Functions Upstream of Both ARL1 and ARL5 to Coordinate the Recruitment of Distinct Classes of Tethering Factors to the TGN Despite the importance of GARP in cellular and organismal physiology, until recently it was unclear how it was recruited to the TGN and how its function was coordinated with that of a different class of tethering factors, long coiled-coil tethers of the golgin family. The golgins mediate long-distance capture of endosome-derived transport carriers, whereas GARP promotes SNARE-dependent fusion of the carriers with the TGN. This past year, we discovered that the ARF-like (ARL) GTPase ARFRP1 is an upstream activator of two other ARL GTPases, ARL1 and ARL5, which in turn recruit golgins and GARP, respectively, to the TGN. In addition, we found that this GTPase cascade is essential for the delivery of retrograde cargos to the TGN. From these findings we concluded that ARFRP1 is a master regulator of retrograde-carrier tethering to the TGN. This mechanism involving the recruitment of distinct classes of tethering factors by a bifurcated GTPase cascade may be paradigmatic of other vesicular fusion events that take place within the cell.